Macular degeneration is a clinical term that is used to describe a variety of diseases that are all characterized by a progressive loss of central vision associated with abnormalities of Bruch's membrane, the neural retina and the retinal pigment epithelium. These disorders include very common conditions that affect older subjects (age-related macular degeneration or AMD) as well as rarer, earlier-onset dystrophies that in some cases can be detected in the first decade of life. Other maculopathies include North Carolina macular dystrophy (Small, et al., 1993), Sorsby's fundus dystrophy (Capon, et al., 1989), Stargardt's disease (Parodi, 1994), pattern dystrophy (Marmor and Byers, 1977), Best disease (Stone, et al., 1992), dominant drusen (Deutman and Jansen, 1970), and radial drusen (“malattia leventinese”) (Heon, et al., 1996).
Histopathologic studies have documented significant and widespread abnormalities in the extracellular matrices associated with the RPE, choroid, and photoreceptors of aged individuals and of those with clinically-diagnosed AMD (Sarks, 1976; Sarks, et al., 1988; Bird, 1992a; van der Schaft, et al., 1992; Green and Enger, 1993; Feeney-Burns and Ellersieck, 1985; Young, 1987; Kincaid, 1992). The most prominent extracellular matrix (ECM) abnormality is drusen, deposits that accumulate between the RPE basal lamina and the inner collagenous layer of Bruch's membrane (FIG. 1).
A number of studies have demonstrated that the presence of macular drusen is a strong risk factor for the development of both atrophic and neovascular AMD (Bressler, et al., 1994; Bressler, et al., 1990; Macular Photocoagulation Study). Drusen causes a lateral stretching of the RPE monolayer and physical displacement of the RPE from its immediate vascular supply, the choriocapillaris. This displacement creates a physical barrier that may impede normal metabolite and waste diffusion between the choriocapillaris and the retina. It is likely that wastes may be concentrated near the RPE and that the diffusion of oxygen, glucose, and other nutritive or regulatory serum-associated molecules required to maintain the health of the retina and RPE are inhibited. It has also been suggested that drusen perturb photoreceptor cell function by placing pressure on rods and cones (Rones, 1937) and/or by distorting photoreceptor cell alignment (Kincaid, 1992).
The complement system consists of a group of globulins in the serum of humans (Hood, L. E. et al. 1984, Immunology, 2d Edition, The Benjamin/Cummings Publishing Co., Menlo Park, Calif., p. 339; See also, U.S. Pat. Nos. 6,087,120 and 5,808,109). Complement activation plays an important role in the mediation of immune and allergic reactions (Rapp, H. J. and Borsos, T., 1970, Molecular Basis of Complement Action, Appleton-Century-Crofts (Meredith), N.Y.). The activation of complement components leads to the generation of a group of factors, including chemotactic peptides that mediate the inflammation associated with complement-dependent diseases. The activities mediated by activated complement proteins include lysis of target cells, chemotaxis, opsonization, stimulation of vascular and other smooth muscle cells, degranulation of mast cells, increased permeability of small blood vessels, directed migration of leukocytes, and activation of B lymphocytes, macrophages and neutrophils (Eisen, H. N., 1974, Immunology, Harper & Row, Publishers, Inc., Hagerstown, Md., p. 512).
There are three major pathways of complement activation. First, the “classical pathway,” which is activated by antibody/antigen binding. Second, the “lectin pathway” or “collecting pathway,” is activated by the binding of acute phase reactant mannose-binding protein (MBP; or mannose-binding lectin, MBL) to a complex carbohydrate. Third, the “alternative pathway,” which involves the recognition of certain polysaccharides (e.g., on microbial surface) and is activated by the presence of a specific substrate called C3bB, a complex of complement proteins. See, e.g., Cooper, Adv Immunol, 37(-HD-):151-216, 1985; Fearon & Austen, J. Exp. Med. 146: 22-33, 1977; Pangburn et al., 266: 16847-53, 1991; Matsushita et al., Microbiol Immunol, 40(12):887-93, 1996; and Turner et al., Res Immunol, 147(2):110-5, 1996. The major classical pathway components are designated C1q, C1r, C1s, C4, C2, C3, C5, C6, C7, C8, C9. The main alternative pathway components are designated Factor B, Factor D, Properdin, H and I. In addition to MBL, the lectin pathway components also include MASP-1 and MASP-2 (Thiel et al., Nature, 386:506-10, 1997). It is also known that more than one pathway can be involved in a single disease process, as in Alzheimer's disease (Akiyama et al., Neurobiol Aging, 21:383-421 2000).
Initiation of the classical pathway begins with antibody binding to a specific antigen. C1q binds the altered Fc region of IgG or IgM that has bound antigen. Upon binding, C1r activates C1s which initiates the activation unit by cleaving a peptide from both C4 and C2. C1s thus cleaves C4 into C4a and C4b and C2 into C2a and C2b. C2a binds to C4b forming C4b2a. C4b2a, the C3 convertase, is a proteolytic enzyme. It cleaves C3 into C3b, which may bind to the activating surface, and C3a which is released into the fluid phase. C3 convertase has the ability to cleave many C3 molecules. This could result in the deposition of a large number of C3b molecules on the activating surface. However, due to the labile nature of C3b, very few molecules actually bind. C4b2a3b, the C5 convertase, is formed when C3 is cleaved. C5 convertase, also an enzyme, can cleave many C5 molecules into C5a and C5b.
The alternative pathway provides natural, non-immune defense against microbial infections. In addition, this pathway amplifies antibody-antigen reactions. Alternative pathway recognition occurs in the presence of C3b and an activating substance such as bacterial lipoprotein, surfaces of certain parasites, yeasts, viruses and other foreign body surfaces, such as biomaterials. C3b originates from classical pathway activation and/or from natural spontaneous hydrolysis of C3. The resulting C3b binds to the surface of the activating substance. In the presence of magnesium, Factor B binds to the C3b which is bound to the activating surface. Factor D then cleaves B, releasing the Ba fragment and forming C3bBb. Properdin stabilizes the C3bBb complex and protects it from decay. C3bBbP is the alternative pathway convertase. It also has the ability to cleave many C3 molecules. Cleavage of C3 results in the formation of C3bBb3b, the C5 convertase. This enzyme is also stabilized by P to form C3bBb3bP. C5 convertase can cleave many molecules of C5 into C5a and C5b.
Binding of MBL to carbohydrates triggers the lectin pathway. MBL is structurally related to the complement C1, C1q, and seems to activate the complement system through an associated serine protease known as MASP-1 or p100, which is similar to C1r and C1s of the classical pathway. MBL binds to specific carbohydrate structures found on the surface of a range of microorganisms, including bacteria, yeasts, parasitic protozoa and viruses, and exhibits antibacterial activity through killing mediated by the terminal, lytic complement components or by promoting phagocytosis. The level of MBL in plasma is genetically determined, and deficiency is associated with frequent infections in childhood, and possibly also in adults. In addition, a further MBL-associated serine protease (MASP-2) was identified which shows a striking homology with the previously reported MASP (MASP-1) and the two C1q-associated serine proteases C1r and C1s (see, e.g., Thiel et al., Nature, 386:506-10, 1997).
The membrane attack complex C5b-9 (also termed complement terminal complex, MAC, or SC5b-9) is common to the complement pathways (see, e.g., Morgan, Crit Rev Immunol, 19(3):173-98, 1999). It begins with the cleavage of C5 by C5 convertase generated during either classical or alternative pathway activation. When C5 is cleaved, C5a is released into the fluid phase while C5b attaches to the activating surface at a binding site distinct from that of C3b. One molecule each of C6 and C7 binds to C5b to form a stable trimolecular complex to which C8 binds. Then, up to 6 molecules of C9 can bind to C8 enhancing the effectiveness of the attack complex to induce membrane damage if the activating surface is a microorganism.
The significance of complement activation is not limited to membrane damage resulting from the attack complex. The active peptides released in the course of complement activation contribute to the immune response by increasing vascular permeability and contraction of smooth muscle, promoting immune adherence, granulocyte and platelet aggregation, enhancing phagocytosis, and directing the migration of neutrophils (PMN) and macrophages to the site of inflammation.
The cleavage of C3 and C5 results in the release of two small biologically active peptides, C3a and C5a. The peptides act as anaphylatoxins. They amplify the immune response by causing the release of histamine, slow releasing substance of anaphylaxis (SRS-A), and heparin from basophils and mast cells. These substances increase capillary permeability and contraction of smooth muscle resulting in edema and inflammation.
In addition to its role as an anaphylatoxin, C5a is a potent chemotactic factor. This mediator causes the directed migration of leukocytes including dendritic cells and monocytes to the site of inflammation so these leukocytes will phagocytize and clear immune complexes, bacteria and viruses from the system.
In a process known as immune adherence, C3b or C4b deposited on a soluble immune complex or surface permit binding of complement receptors on PMN, macrophages, red blood cells and platelets. In these cases C3b and C5b are considered opsonins as their presence results in more effective phagocytosis.
New diagnostics and therapeutics for macular degeneration-related disorders are needed. For example, there is currently no reliable biochemical or genetic means in routine use for diagnosing, e.g., AMD. In addition, there is no therapy currently in use that significantly slows the degenerative progression of AMD for the majority of subjects. Current AMD treatment is limited to laser photocoagulation of the subretinal neovascular membranes that occur in 10-15% of affected subjects. The latter may halt the progression of the disease but does not reverse the dysfunction, repair the damage, or improve vision.